EP3107996B1 - Cellules tscm et leurs procédés d'utilisation - Google Patents

Cellules tscm et leurs procédés d'utilisation Download PDF

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EP3107996B1
EP3107996B1 EP15751942.2A EP15751942A EP3107996B1 EP 3107996 B1 EP3107996 B1 EP 3107996B1 EP 15751942 A EP15751942 A EP 15751942A EP 3107996 B1 EP3107996 B1 EP 3107996B1
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cells
cell
dcs
antigen
patient
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EP3107996A4 (fr
EP3107996A1 (fr
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Mark Debenedette
Charles Nicolette
Joseph HORVATINOVICH
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Colmmune Inc
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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    • A61K35/14—Blood; Artificial blood
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
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    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
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    • A61K39/46449—Melanoma antigens
    • A61K39/464491—Melan-A/MART
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01—MEASURING; TESTING
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to an in vitro method for obtaining an autologous population of cells enriched for T SCM cells suitable for introduction into a human patient.
  • the present disclosure also relates to T SCM cells and uses thereof T SCM cells can be used to help identify and treat patients who are likely to experience particular treatment outcomes.
  • Cell therapy utilizes modified antigen presenting cells (APCs) or immune effector cells to initiate an immune response in a patient.
  • APCs modified antigen presenting cells
  • Antigen presenting cells are central to cell therapy because they initiate the immune response; specifically, they are capable of inducing a primary immune response from T lymphocytes.
  • DCs Dendritic cells
  • APCs Dendritic cells
  • CTL cytotoxic T lymphocyte
  • immature DCs that reside in peripheral tissues are equipped to capture antigens and to produce immunogenic MHC-peptide complexes.
  • maturation-inducing stimuli such as inflammatory cytokines
  • immature DCs develop into potent T cell stimulators by upregulating adhesion and costimulatory molecules and migrate into secondary lymphoid organs to select and stimulate rare antigen-specific T cells.
  • potent stimulation of T cells occurs only after DC maturation, a process that increases the availability of MHC/peptide complexes on the cell surface in addition to co-stimulatory molecules that direct the effector function of the responding T-cells.
  • Co-stimulation is typically necessary for a T cell to produce sufficient cytokine levels that induce clonal expansion.
  • dendritic cells that make them potent antigen presenting cells is that they are rich in co-stimulatory molecules of the immune response, such as the molecules CD80 and CD86, which activate the molecule CD28 on T lymphocytes.
  • T-helper cells express CD40L, which ligates CD40 on DCs.
  • DCs Another special characteristic of DCs is to deploy different functions depending on their stage of differentiation.
  • the capture of antigen and its transformation are the two principal functions of the immature dendritic cell, whereas its capacities to present the antigen in order to stimulate the T cells increases as the DCs migrate into the tissues and the lymphatic ganglia.
  • This change of functionality corresponds to a maturation of the dendritic cell.
  • the passage of the immature dendritic cell to the mature dendritic cell represents a fundamental step in the initiation of the immune response. Traditionally, this maturation was followed by monitoring the change of the surface markers on the DCs during this process.
  • Some of the more important cell surface markers characteristic of the different stages of maturation of the dendritic cells include CD34+ for hematopoietic stem cell; CD14++, DR+, CD86+, CD16+/-, CD54+, and CD40+for monocytes; CD14+/-, CD16-, CD80+/-, CD83-, CD86+, CD1a+, CD54+, DQ+, DR++ for immature dendritic cells, and CD14-, CD83++, CD86++, CD80++, DR+++, DQ++, CD40++, CD54++, CD1a +/- for mature dendritic cells, where "+” indicates positive expression, "++” indicates higher expression, "+/-” indicates weaker expression, and "-” indicates very weak or undetectable expression.
  • the surface markers can vary depending upon the maturation process.
  • Mature DCs are currently preferred to immature DCs for immunotherapy. Only fully mature DC progeny lack GM-CSF Receptor (GM-CSF-R) and remain stably mature upon removal and/or in the absence of GM-CSF. Also, mature DCs have been shown to be superior in inducing T cell responses in vitro and in vivo. Mature dendritic cells also are useful to take up and present antigen to T-lymphocytes in vitro or in vivo. The modified, antigen presenting DCs and/or T cells educated from these modified DCs have many applications, including diagnostic, therapy, vaccination, research, screening and gene delivery.
  • GM-CSF-R GM-CSF Receptor
  • T SCM cells comprising activating T-lymphocytes with anti-CD3/CD28-conjugated magnetic beads and then culturing in the presence of IL-1 and IL-15 has been described in Gattinoni, et al., 2011. Gattinoni, et al. 2010 also describes that infusion of T SCM cells in immunodeficient mice induces an immune response.
  • the generation of human T SCM cells by activating T-cells with ⁇ -CD3/CD28 beads in the presence of the GSK-3 ⁇ inhibitor TWS119 is described in Cieri et al., 2013. US 2013/280805 describes a method for producing PME-CD40L mature DCs.
  • PME-CD40L DCs can be used to treat a human patient having an immune disease or disorder and also to stimulate the production in vivo of advantageous T cells.
  • PME-CD40L DCs are produced by methods previously developed and described in WO2006042177 (Healey et al. ); WO2007117682 (Tcherepanova et al. ); DeBenedette et al. (2008) J. Immunol. 181: 5296-5305 ; and Calderhead et al. (2008) J. Immunother. 31: 731-41 .
  • PME-CD40L DCs can be produced, for example, by a method comprising the sequential steps of: (a) culturing isolated immature dendritic cells (iDCs) with an interferon gamma receptor (IFN- ⁇ R) agonist in the presence of a TNF- ⁇ R agonist and PGE 2 for approximately 12 to 30 hours to produce CD83 + mature dendritic cells; and (b) transfecting said CD83 + mature dendritic cells (mDCs) with a CD40 agonist to produce a transient CD40 signal.
  • the CD40 agonist is mRNA encoding a CD40L polypeptide.
  • the mRNA encodes a CD40L polypeptide consisting of amino acid residues 21-261 of SEQ ID NO:2 of WO2007117682 .
  • the mRNA encoding the CD40L polypeptide may be cotransfected with an mRNA encoding an antigen.
  • PME-CD40L DCs are mature DCs that are also phenotypically CD83 + and CCR7 + .
  • T SCM cells are stem cell memory T cells that are multipotent and can also give rise to progeny cells that are themselves T SCM cells, which makes possible the generation of additional T SCM cells.
  • the production of T SCM cells by exposure to PME-CD40L DCs can occur in vivo in human patients having immune diseases or disorders, including AIDS or infection with HIV, and can serve as an indicator of a patient's immune response and thus clinical prognosis.
  • PME-CD40L DCs can also induce T SCM cells in vitro by coculturing PME-CD40L DCs with lymphocytes. This coculturing of PME-CD40L DCs with lymphocytes results in a cell population enriched for T SCM cells. These cells can then be reintroduced into a patient to help stimulate the immune response of the patient from whom they were derived ( i.e., autologous treatment), and can also be used, for example, to treat another patient in adoptive transfer therapy (i.e., heterologous treatment). In some examples, the T SCM cells are further purified from the lymphocyte population or are further enriched prior to use in adoptive transfer therapy.
  • the present invention relates to an in vitro method for obtaining an autologous population of cells enriched for T SCM cells suitable for introduction into a human patient comprising the steps of:
  • the PME-CD40L mature DCs are loaded with an antigen.
  • the DCs are loaded with said antigen by transfection with RNA encoding said antigen.
  • the RNA is prepared from cells of an HIV-infected patient.
  • the antigen is prepared from cancer cells of a cancer patient.
  • the T SCM cells are CD8+, CD95+, CD28+, CCR7+, and CD45RA+.
  • the T SCM cells are enriched as cells positive for CD27, CD28, and CD45RA.
  • the method further comprises combining said T SCM cells with a pharmaceutically acceptable carrier.
  • immature DCs are sequentially signaled with a first signal (an IFN- ⁇ receptor agonist and/or a TNF- ⁇ receptor agonist) to produce CD83 + CCR7 - mature DCs and then are signaled with a second signal (a CD40 agonist), producing CD83 + CCR7 + mature DCs; various IFN- ⁇ receptor agonists and/or TNF- ⁇ receptor agonists may be used.
  • a first signal an IFN- ⁇ receptor agonist and/or a TNF- ⁇ receptor agonist
  • a second signal a CD40 agonist
  • immature DCs are first phenotypically matured by adding 'inflammatory mediators' IFN- ⁇ and TNF- ⁇ to the culture medium; optionally, PGE 2 is also added. Then, approximately 12-30 hours later (in some examples about 18 hrs later), the cells are electroporated with CD40L mRNA and, optionally, antigen-encoding mRNA. This 'PME-CD40L process' produces CD83 + CCR7 + mature DCs. Cells harvested from this process after electroporation ( e.g., 4 hrs post electroporation) and formulated as a vaccine were shown to mediate maximum immunopotency in in vitro assays.
  • PME-CD40L process for Post Maturation Electroporation with CD40L
  • PME-CD40L DCs Dendritic cells made by the PME-CD40L process
  • PME-CD40L DCs are phenotypically different than previously known dendritic cells.
  • PME-CD40L DCs can support long term antigen-specific CTL effector function and induce a type of effector memory CTLs designated “Rapidly Expanding High-Avidity” (“REHA”) cells (see DeBenedette et al. (2008) J. Immunol. 181: 5296-5305 ). These cells retain the capacity to expand, produce cytokines, and kill target cells--all critical events in mediating robust long-term CTL effector function.
  • REHA Rapidly Expanding High-Avidity
  • PME-CD40L DCs were shown to preferentially induce a population of CD28 + CD45RA memory/effector T cells from a population of antigen-specific T cells, which could be either naive T cells or antigen-experienced T cells.
  • the resulting effector/memory T cells produced IFN ⁇ and IL-2 and could kill target cells; thus, these cells differed from both effector T cells (which produce IFN ⁇ and can kill target cells, but do not produce IL-2) and memory T cells (which produce IFN ⁇ and IL-2, but do not kill target cells).
  • PME-CD40L DCs are potent immunostimulatory agents that are useful in producing T SCM cells and thus are useful in methods of the invention.
  • Methods for producing PME-CD40L DCs comprise the sequential steps of: (a) signaling isolated immature dendritic cells (iDCs) with a first signal comprising an interferon gamma receptor (IFN- ⁇ R) agonist, and optionally a TNF- ⁇ R agonist, to produce IFN- ⁇ R agonist signaled dendritic cells; and (b) signaling said IFN- ⁇ R agonist signaled dendritic cells with a second transient signal comprising an effective amount of a CD40 agonist to produce CD83 + CCR7 + mature dendritic cells.
  • IFN- ⁇ R interferon gamma receptor
  • the CD83 + CCR7 + mature DCs transiently express CD40L polypeptide; in some instances, the CD40L is predominantly localized intracellularly rather than on the cell surface. At least 60%, at least 70%, at least 80% or at least 90% of the CD40L polypeptide may be localized intracellularly.
  • PME-CD40L DCs exhibit some distinctive characteristics, including: (i) they demonstrate elevated cell surface expression of co-stimulator molecules CD80, CD83, and CD86; ii) they are CCR7 + ; and iii) they secrete IL-12 p70 polypeptide or protein, and/or secrete significantly reduced levels (0 to 500 pg per ml per million DCs) of IL-10 (see, e.g., data and experiments presented in WO2006042177 (Healey et al. ) and WO2007117682 (Tcherepanova et al. )).
  • CD83 + CCR7 + DCs produce at least 1000 pg IL-12 per 10 6 DCs; IL-10 and IL-12 levels can be determined by ELISA of culture supernatants collected at up to 36 hrs post induction of DC maturation from immature DCs ( Wierda et al. (2000) Blood 96: 2917 ; Ajdary et al. (2000) Infection and Immunity 68: 1760 ).
  • PME-CD40Ls have been produced by sampling a cell or small (sub)population of DCs from a cell population for the presence of mature DCs expressing CD40L mRNA and/or CD40L polypeptide, or expressing interleukin 12 (IL-12) p35 protein.
  • Other characteristics of these cells are discussed, for example, in WO2006042177 (Healey et al. ); WO2007117682 (Tcherepanova et al. ); DeBenedette et al. ((2008) J. Immunol. 181: 5296-5305 ); and Calderhead et al. ((2008) J. Immunother. 31: 731-41 ).
  • Immature DCs used to produce PME-CD40L DCs can be isolated or prepared from a suitable tissue source containing DC precursor cells and differentiated in vitro to produce immature DCs.
  • a suitable tissue source can be one or more of: bone marrow cells; peripheral blood progenitor cells (PBPCs); peripheral blood stem cells (PBSCs); and cord blood cells.
  • the tissue source is a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the tissue source can be fresh or frozen, and can be pre-treated with an effective amount of a growth factor that promotes growth and differentiation of non-stem or progenitor cells, which are then more easily separated from the cells of interest.
  • the immature DCs can also be isolated from peripheral blood mononuclear cells (PBMCs) which optionally are treated with an effective amount of granulocyte macrophage colony stimulating factor (GM-CSF) in the presence or absence of interleukin 4 (IL-4) and/or IL-13, so that the PBMCs differentiate into immature DCs.
  • PBMCs are cultured in the presence of GM-CSF and IL-4 for about 4-7 days, preferably about 5-6 days, to produce immature DCs.
  • the first signal is given at day 4, 5, 6, or 7, and most preferably at day 5 or 6.
  • GM-CSF as well as IL-4 and/or IL-13 may be present in the medium at the time of the first and/or second signaling.
  • hematopoietic factor to be administered may be determined by one skilled in the art by monitoring the frequency of cell types in individuals to whom the factor is being administered.
  • U.S. Patent No. 6,475,483 teaches that dosages of G-CSF of 300 micrograms daily for 5 to 13 days and dosages of GM-CSF of 400 micrograms daily for 4 to 19 days result in significant yields of dendritic cells.
  • the immature dendritic cells can be signaled with an effective amount of a TNF- ⁇ receptor agonist followed by signaling with a CD40 agonist.
  • the immature DCs may be contacted with PGE 2 at about the same time that they receive the first signal of an IFN- ⁇ R agonist and a TNF- ⁇ R agonist.
  • signaling is in the absence of an effective amount of IL-1 ⁇ and/or IL-6.
  • GM-CSF and at least one of IL-4 or IL-13 may be present in the medium at the time the dendritic cells receive the first and second signals.
  • IFN- ⁇ receptor agonists can be accomplished by contacting a cell directly with IFN- ⁇ polypeptides and/or proteins and/or TNF- ⁇ polypeptides or proteins and/or CD40 agonists, respectively.
  • IFN- ⁇ and TNF- ⁇ receptor agonists can be aptamers, antibodies, and the like, that have a similar biological activity.
  • signaling of a cell with IFN- ⁇ R agonists, TNF- ⁇ R agonists and/or CD40 agonists can occur upon translation of mRNA encoding such polypeptides or proteins within the dendritic cell.
  • Such mRNA may be introduced into the cell by transfection or other means, and the signaling then occurs upon expression of the IFN- ⁇ R agonist, TNF- ⁇ R agonist and CD40 agonist polypeptides and/or proteins.
  • signaling can be initiated by providing the signaling agonist in the culture medium, introduction of the agonist into the cell, and/or upon translation within the dendritic cell of an mRNA encoding an agonistic polypeptide.
  • the methods can be practiced in vivo or ex vivo. Dendritic cells matured ex vivo according to the methods described herein can then be administered to the subject to induce or enhance an immune response along with the T SCM s produced by coculturing with the DCs.
  • Dendritic cells can be further modified by the administration of an immunogen (e.g., an antigen) to the DCs.
  • the immunogen can be delivered in vivo or ex vivo.
  • the immunogen can be delivered to the cells using methods known in the art, and can be delivered as polypeptides or proteins ( e.g., by "pulsing") or as nucleic acids encoding the immunogen ( e.g, by transfection or electroporation).
  • the polynucleotide is an mRNA.
  • the antigen-encoding mRNA is electroporated together with an mRNA encoding a CD40 agonist or substantially concurrent with CD40 agonist signaling.
  • PME-CD40L DCs can also be transfected with RNA encoding antigens from any pathogen or disease of interest; such antigens can be from one individual subject or multiple subjects and can be from a pathogen infection of the subject from which the antigens are isolated or from another subject. Consensus antigens and pathogen-specific antigens are known in the art and may also be used in these methods of preparing PME-CD40L DCs.
  • the DCs will process the antigens and display the antigens on their cell surface; these mature DCs can be used to educate naive immune effector cells.
  • RNA encoding antigens from a cancer and/or tumor sample removed from a subject may be used to transfect DCs in this manner.
  • RNA encoding HIV antigens from a sample removed from a subject may also be used to transfect DCs.
  • PME-CD40L DCs that were transfected with MART-encoding mRNA stimulated autologous CD8+ T cells to produce responder CD8+ T cells, as described, for example, in WO2006042177 (Healey et al. ) and WO2007117682 (Tcherepanova et al. ) .
  • PME-CD40L matured DCs loaded with total amplified Renal Cell Carcinoma ("RCC") tumor RNA induced a fully autologous CTL response (see WO2006042177 (Healey et al. )).
  • RRCC Renal Cell Carcinoma
  • the PME-CD40L DCs used in methods described herein to produce T SCM cells are transfected with RNA encoding part or all of the HIV proteins Gag, Nef, Tat, and Rev, as described in WO2006031870 and U.S. Pub. No. 20080311155 (Nicolette et al. ) .
  • DCs are transfected with RNA encoding one or more polypeptides from multiple strains of HIV present in an individual subject; the RNA is derived from nucleic acid amplification of pathogen polynucleotides.
  • Primers to amplify such pathogen polynucleotides can be designed to compensate for sequence variability between multiple strains of said pathogen, for example, when said pathogen is HIV, as described in WO2006031870 and U.S. Pub. No. 20080311155 .
  • Such primers can include, for example, primers disclosed in WO2006031870 , including forward and reverse primers for Gag, Nef, Tat, and Rev.
  • the DCs resulting from this process have been shown to be capable of stimulating an immune response to HIV in HIV patients. In this manner, a DC vaccine autologous to a patient can be produced and used to stimulate an immune response to the HIV strains found in that patient.
  • PME-CD40L DCs can also be stored by contacting an enriched dendritic cell population with a suitable cryopreservative under suitable conditions and frozen (see, e.g., WO 2002016560 and U.S. Pat. No. 8,574,901 (Schuler et al. )).
  • CD34 + stem cells can be isolated from bone marrow cells or by panning the bone marrow cells or other sources with antibodies which bind unwanted cells, such as CD4 + and CD8 + (T cells) (see, e.g., Inaba, et al. (1992) J. Exp. Med. 176: 1693-1702 ).
  • Human CD34 + cells can be obtained from a variety of sources, including cord blood, bone marrow explants, and mobilized peripheral blood. Purification of CD34 + cells can be accomplished by antibody affinity procedures, for example, as described in Paczesny et al. (2004) J. Exp. Med. 199: 1503-11 ; Ho et al. (1995) Stem Cells 13 (suppl. 3): 100-105 ; Brenner (1993) Journal of Hematotherapy 2:7-17 ; and Yu, et al. (1995) PNAS 92: 699-703 .
  • CD34 + stem cells can be differentiated into dendritic cells by incubating the cells with appropriate cytokines, as is known in the art.
  • human CD34 + hematopoietic stem cells can be differentiated in vitro by culturing the cells with human GM-CSF and TNF- ⁇ (see, e.g., Szabolcs, et al. (1995) J. Immunol. 154: 5851-5861 ).
  • SCF or other proliferation ligand e.g., F1t3
  • Dendritic cells can be isolated by florescence activated cell sorting (FACS) based on expression of cell surface markers or by other standard methods.
  • FACS florescence activated cell sorting
  • DCs can be generated from non-proliferating CD14 + precursors (monocytes) in peripheral blood by culture in medium containing GM-CSF and IL-4 or GM-CSF and IL-13 (see, e.g., WO 97/29182 ; Sallusto and Lanzavecchia (1994) J. Exp. Med. 179: 1109 and Romani et al. (1994) J. Exp. Med. 180:83 ). In some instances, patients can be pretreated with cytokines such as G-CSF, but in most cases this is not necessary because CD14 + precursors are sufficiently abundant ( Romani et al. (1996) J. Immunol. Methods 196: 137 ).
  • DCs used to produce T SCM cells in methods described herein are derived from the same patient or subject; that is, the DCs and the T SCM cells or their precursor cells are obtained from the same patient or subject ( i.e., they are autologous). In other examples, the DCs and the T SCM cells are derived from different subjects ( i.e., they are allogeneic or heterologous).
  • T cells are isolated from mammals and cocultured with PME-CD40L DCs in vitro to produce T SCM cells.
  • established procedures are used to separate PBMCs from red blood cells and neutrophils with Ficoll-Hypaque density gradient centrifugation.
  • Cells are washed with modified AIM-V (which consists of AIM-V (GIBCO® Life Technologies) with 2 mM glutamine, 10 ⁇ g/ml gentamicin sulfate, 50 ⁇ g/ml streptomycin) supplemented with 1% fetal bovine serum (FBS).
  • AIM-V which consists of AIM-V (GIBCO® Life Technologies) with 2 mM glutamine, 10 ⁇ g/ml gentamicin sulfate, 50 ⁇ g/ml streptomycin
  • T cells are enriched by negative and/or positive selection with appropriate monoclonal antibodies coupled to columns or magnetic beads according to standard techniques and/or manufacturer or provider directions.
  • An aliquot of cells is analyzed for cell surface phenotype including CD4, CD8, CD3 and CD14, and for the purpose of illustration only, cells are washed and resuspended at a concentration of about 5 x 10 5 cells per ml of AIM-V modified as above and containing 5% FBS and 100 U/ml recombinant IL-2 (rIL-2) (referred to as "supplemented AIM-V").
  • the term "coculture” refers to a cell culture known to contain at least two different types of cells.
  • HIV-infected cells When cells are isolated from an HIV patient, HIV-infected cells may be preferentially removed from the population using reagents such as, for example, CD4-PE40 (e.g., at 25nM).
  • CD4-PE40 is a recombinant protein consisting of the HIV-1-binding CD4 domain linked to the translocation and ADP-ribosylation domains of Pseudomonas aeruginosa exotoxin A; it has been shown to inhibit p24 production in HIV-infected cell cultures and to selectively kill HIV-1-infected cells.
  • OKT3 monoclonal antibody Order to stimulate cell proliferation.
  • T SCM cells can be isolated from patient material; for example, T SCM CTLs can be isolated from peripheral blood or tumor infiltrating lymphocytes.
  • T cells or PBMCs are isolated from human patients and cocultured with PME-CD40L DCs derived from the same patient or subject to produce and/or expand T SCM cells in vitro.
  • These T SCM CTLs can then be used for adoptive transfer therapy by infusing them back into the same patient (autologous therapy) or into another patient (allogeneic therapy).
  • Successful allogeneic adoptive transfer therapy of hematopoietic stem cells and lymphocytes has been reported, for example, in Cieri et al. ((2014) Immunol. Rev. 257: 165-180 ) and Kolb et al. ((1995) Blood 86: 2041-50 ).
  • T SCM CTLs can be identified in co-cultures of T-cells and PME-CD40L DCs and isolated and/or expanded in vitro. If PME-CD40L DCs are loaded with antigen and used to expand a population of T SCM cells, the resulting population will include T SCM cells that are reactive to the antigen (for example, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the T SCM cells produced by the expansion will be reactive to the antigen). This is demonstrated, for example, by the data provided in Figure 1 and discussed in Example 2, which illustrates the ability of PME-CD40L DCs loaded with the MART-1 tumor antigen to expand a population of MART-1-reactive T SCM CTLs.
  • T SCM CTLs can be produced that are reactive to any particular antigen or antigens; these can be used to enhance or stimulate the patient's immune response to the antigen by adoptive transfer therapy.
  • antigens can be prepared from a patient's own cancer cells and loaded into DCs that are used to expand a population of T SCM CTLs that are then infused back into the patient.
  • antigens are prepared from an HIV patient ( i.e., a patient infected with HIV) and loaded into DCs that are used to expand a population of T SCM CTLs that are infused back into the patient.
  • HIV patient i.e., a patient infected with HIV
  • DCs that are used to expand a population of T SCM CTLs that are infused back into the patient.
  • T SCM CTLs may also produce various cytokines, such as, for example, TNF- ⁇ or IFN- ⁇ .
  • cytokines such as, for example, TNF- ⁇ or IFN- ⁇ .
  • the data shown in Figure 1 revealed that some of the MART-1+ T SCM CTLs were "multi-functional," with 1.8% expressing TNF- ⁇ , 3.2% expressing CD107a, and 1.5% expressing IFN- ⁇ ; IL-2 expression was not detected.
  • Methods are known in the art to separate cells based on particular functional attributes such as their expression of specific cytokines (e.g., as discussed in Kammula et al. (1999) J. Immunol. 12: 6867-75 and Kammula et al. (2008) J. Transl. Med.
  • cells can be selected on the basis of cytokine expression using a cytokine capture reagent (e.g., as discussed in Brosterhus et al. (1999) Eur. J. Immunol. 12: 4053-59 ).
  • a cytokine capture reagent e.g., as discussed in Brosterhus et al. (1999) Eur. J. Immunol. 12: 4053-59 ).
  • cell surface markers can be used to identify and/or isolate cells for various purposes.
  • DCs can be distinguished from other cells because they express MHC molecules and costimulatory molecules (e.g., B7-1 and B7-2) and lack markers specific for granulocytes, NK cells, B cells, and T cells.
  • T SCM CTLs have some markers in common with naive cells such as, for example, CD27, CD28, and CD45RA. However, since T SCM cells have been exposed to antigen they also express activation markers such as, for example, CD95 and CD122.
  • T SCM cells can be CD4+ or CD8+, and can also be CCR7+.
  • T SCM cells facilitates identification, purification, and separation of these cells from other cells expressing at least one different marker; any suitable combination of markers may be used and is readily determined by one of skill in the art. Negative marker or cell selection may also be used.
  • the disclosure provides methods of identifying and/or separating, isolating, or enriching T SCM cells from other cells on the basis of expression of one or more of CD27, CD28, CD45RA, CD95, CD122, CD4, CD8, CCR7, and PD-1.
  • T SCM cells enriched as cells that are positive for CD27, CD28, and CD45RA.
  • T SCM cells are further enriched as positive for CD8, CD95, CD28, CD7, and CD45RA.
  • T SCM cells may also be identified and/or separated or enriched from other cells based on their expression of PD-1.
  • Cells can be isolated and/or characterized by flow cytometry methods such as FACS analysis as well as by any suitable method known in the art, including but not limited to methods such as column chromatography, Western blots, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
  • flow cytometry methods such as FACS analysis as well as by any suitable method known in the art, including but not limited to methods such as column chromatography, Western blots, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperd
  • Labeling agents which can be used to label cell antigens (including cell surface markers) include but are not limited to monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any known method, such as immunoblotting, Western blot analysis, tracking of radioactive or bioluminescent markers, capillary electrophoresis, or other methods which track a molecule based on size, charge or affinity.
  • T SCM CTLs can be identified by multi-color flow cytometry as cells that are positive for ( i.e., express at detectable levels) the markers CD8, CD95, CD28, CCR7, and CD45RA (also designated "CD8+/CD95+/CD28+/CCR7+/CD45RA+").
  • T SCM CTLs can also be enriched, isolated or purified from other cells using magnetic bead isolation of cells having one or more of these markers, or in some instances it may be preferable to remove other cell types from a population including T SCM cells using appropriate markers.
  • Cell separation methods based on the expression of surface markers are known in the art and include the use of magnetic bead isolation, FACS sorting (e.g., as discussed in Basu et al. (2010) J. Vis. Exp. 41 ), and microelectromechanical systems chips (“MEMS" chips)-based sorting (e.g., as discussed in Shoji and Kawai (2011) Top. Curr. Chem. 2011: 1-25 ).
  • FACS machines and cell sorters are commercially available (e.g., the BD Bioscience LSRII and the BD FACSAria) and can be used according to manufacturer's instructions.
  • T SCM cells can be isolated or separated from other cells by positive or by negative selection where appropriate, or by both positive and negative selection.
  • T SCM cells can be enriched from a population including other cells such as PBMCs or lymphocytes using negative selection to deplete other cell types followed optionally by positive selection for CD8 and/or CD4 and positive selection for CD95.
  • Kits and reagents are known in the art for a variety of purification steps, allowing one of skill in the art to isolate or purify a known cell type; for example, Invitrogen's Dynabeads® UntouchedTM Human T cell kit is designed to deplete human B cells, NK cells, monocytes, macrophages, platelets, dendritic cells, granulocytes, and erythrocytes using antibodies including mouse IgG antibodies against non-T cells: human CD14, CD16, CD19, CD36, CD56, CDw123, and CD235a. It will be appreciated from this example that one of skill in the art is capable of selecting particular (often commercially-available) antibodies and selection tools to enrich and/or deplete known cell types from a population of cells.
  • Selection for cells bearing particular markers can be performed for one marker at a time or for more than one marker at a time (e.g., as discussed in Stemberger et al. (2012) PLoS One 4:e35798 ). Selection can also be performed serially, and different types of selection can be used on a particular group or population of cells in subsequent selection steps to obtain a desired subpopulation. Cells can also be selected based on their antigen specificity directly by isolating T cells reactive to HLA-peptide complexes (e.g., as discussed in Keenan et al. (2001) Br. J. Haematol. 2: 428-34 ). Cell markers that are useful for identification, screening, and/or selection include CD4, CD8, CD27, CD28, CD38, CD57, PD-1, HLA-DR, CD45RA, and CD95.
  • T SCM CTLs can be expanded in vitro with PME-CD40L DCs presenting the antigen(s) of interest to produce a population of T SCM CTLs reactive to a particular antigen or set of antigens.
  • the expansion of the cells can be performed before or after isolation of the T SCM cells from other cells, or both before and after (so that the T SCM cells are purified or enriched both before the expansion step and after the expansion step).
  • Methods of expanding T cells are known in the art, such as methods making use of IL-15 (see, e.g., Klebanoff et al. (2004) Proc. Nat'l. Acad. Sci. USA 7: 1969-74 ) and IL-21 (see, e.g., Albrecht et al.
  • cell developmental pathway modulators such as, for example, rapamycin can also be used to promote memory CD8+ T cell formation (see, e.g., Rao et al. (2010) Immunity 1: 67-78 ).
  • T cells with particular antigen receptors can also be generated using methods of genetic modification, for example, using lentiviral vectors as described in Wang et al. ((2012) J. Immunother. 35: 689-701 ) and Terakura et al. ((2012) Blood 1: 72-82 ), or chimeric antigen receptors as discussed in Barrett et al. ((2014) Ann. Rev. Med. 65: 333-47 ).
  • lentiviral transduced cells are not suitable for therapeutic purposes, and proviral integration into the genome of the transduced cell can result in leukemia; thus, alternative methods are preferred.
  • AGS-004 refers to PME-CD40L DCs containing "GNVR", the RNA antigen payload encoding the antigens Gag (G), Nef (N), Vpr (V), and Rev (R) (also called “GNVR DCs”) (as described, for example, in WO2006031870 ).
  • AGS-004 was found to increase the number of T SCM cells in treated patients and to stimulate the immune response (see, e.g., Example 3 and Example 5, and data shown in the figures).
  • T SCM CTLs can be detected or identified, for example, in co-cultures containing PBMCs and PME-CD40L DCs in vitro as well as in patient blood or other tissues.
  • multi-color flow cytometry detected T SCM cells among PBMCs of HIV patients treated with PME-CD40L DCs encoding HIV antigens (GNVR DCs).
  • Proliferating T SCM CD4+ and CD8+ T cells were detected in PMBCs collected from HIV subjects receiving AGS-004 after in vitro stimulation with PME-CD40L DCs encoding HIV antigens.
  • Viral load data and immune monitoring data from HIV patients was analyzed to determine the relationship between time to viral rebound after anti-retroviral therapy (ART) interruption and the anti-GNVR T cell response.
  • Subjects on ART have no detectable HIV viral load, but once drug therapy is withdrawn the virus will typically rebound. A delay in viral rebound is indicative of an anti-HIV immune response.
  • CFSE can be used in conjunction with other cell markers to identify cell types that are proliferating.
  • the frequency of CFSElo T cells represents the percentage of T cells proliferating in vitro after restimulation with GNVR DCs.
  • Single time point data for the frequency of CFSElo T cells was analyzed at visit 9 and visit 13 post AGS-004 treatment, but neither time point showed an association between the percentage of proliferating T cells and extended time to viral rebound in the patient or reduction in viral load.
  • the four patients with a favorable response compared to the other three subjects had a marked difference in the percent change of proliferating CD27 + /CD28 + /CD45RA + CD4 T SCM cells ( Figure 2B ) and CD27 + /CD28 + /CD45RA + CD8 T SCM cells ( Figure 2D ) measured from baseline (visit 3) and after administration of AGS-004 determined as the mean response measured by averaging the response detected at visit 9 and visit 13.
  • T SCM cells induced in vivo after treatment with PME-CD40L DC are present in vitro for downstream isolation and expansion.
  • the correlation between T SCM cells and favorable response to AGS-004 makes the frequency and/or change in T SCM cells in a patient a useful indicator of immune response in a patient.
  • the frequency and/or change in T SCM cells in a patient following a treatment is a valuable tool for assessing a patient's likely clinical outcome.
  • T SCM cells disclosed herein have low or no expression of PD-1, a marker associated with dysfunctional CD4 and CD8 T cell activation. In this manner, expression of PD-1 can serve as an indicator of immune response.
  • T SCM cells in a patient By monitoring the frequency and/or change in T SCM cells in a patient (and/or the expression of PD-1 by those cells), it is possible to predict or determine whether a treatment of a patient has been or will be effective in inducing an immune response as measured, e.g., by a decrease in viral load or an extended time to viral rebound. Similarly, by monitoring the frequency and/or change in T SCM cells in a patient, it is also possible to evaluate when a treatment has been effective and/or when a patient has had enough doses of a treatment (such as, for example, AGS-004) to be effective in inducing an immune response.
  • a treatment such as, for example, AGS-004
  • an increase of at least 20%, 30%, 40%, 50%, 60%, 100%, or 200% or more of T SCM cells in a patient will indicate that the patient has had a sufficient immune response that a treatment (e.g., treatment with AGS-004) has reached a treatment threshold and may properly be discontinued.
  • a treatment e.g., treatment with AGS-004
  • T SCM cells in a HIV patient will indicate that STI may continue; that is, that ART is not required for effective treatment.
  • effective treatment may require that the STI end and ART be resumed.
  • Such treatment decisions are within the skill of a clinician with the guidance of known measures of patient health and also by changes in patient T SCM cell level as described herein. In this manner, the present disclosure provides methods of determining whether a treatment has been effective and/or whether a particular treatment should be continued or discontinued.
  • methods of determining or confirming effective treatment of a patient for an immune-related disease or disorder comprise obtaining an aliquot of blood from the patient; quantifying the number of T SCM cells present in the patient's blood; administering a treatment to said patient comprising autologous mature DCs prepared in vitro; quantifying the number of T SCM cells present in the patient's blood; and confirming that the number or frequency of T SCM cells present in the patient's blood has reached a treatment threshold, such as, for example, an increase in at least 50%, 100%, 150%, or 200% of T SCM cells present in the patient's blood, where the treatment threshold indicates that a treatment has been effective and may be discontinued.
  • a treatment threshold such as, for example, an increase in at least 50%, 100%, 150%, or 200% of T SCM cells present in the patient's blood
  • T SCM cells in the patient's blood has not reached the treatment threshhold, additional treatment(s) are indicated, such as, for example, additional doses of AGS-004 and/or the need to resume Anti-Retroviral Therapy ("ART").
  • Other treatment thresholds or measures can also be used, such as, for example, HIV RNA assays.
  • the treatment threshold is an increase in T SCM cells that are proliferating and/or in Tscm cells that have low or no expression of PD-1.
  • an increase in T cells and/or T SCM cells expressing PD-1 indicates that an immune response is defective or ineffective.
  • T SCM cells can serve as an indicator or measure of a patient's immune response.
  • the desired result of a treatment is that the immune response has been stimulated so that an increase in T SCM cells can be measured (that is, it is above the level of detection, such as at least 10%, 20%, or 30% or more); in these examples, a treatment is determined to be effective if it results in such an increase.
  • Also disclosed herein are methods of measuring an immune response in a patient having a disease or disorder comprising the steps of: obtaining a sample of the patient's blood for determining the quantity and/or frequency of T SCM cells present therein; administering autologous mature DCs prepared in vitro to said patient; subsequently obtaining a sample of the patient's blood for determining the quantity and/or frequency of T SCM cells present therein post-treatment; and comparing the quantity and/or frequency of T SCM cells present in the patient's blood post-treatment to the quantity prior to treatment, wherein an increase of Tscm cells indicates that an immune response has been induced in the patient.
  • Also disclosed herein are methods of making a recommendation for treatment of a patient comprising the steps of: quantifying the number of T SCM cells present in a sample of a patient's blood to establish a baseline reading; following administration to said patient of a treatment comprising autologous mature DCs prepared in vitro, quantifying the number of T SCM cells present in a sample of said patient's blood to establish a post-treatment reading; comparing said baseline reading and said post-treatment reading to determine whether the frequency or amount of T SCM cells present in the sample of the patient's blood has increased to meet the treatment threshhold; and making a recommendation to continue treatment of said patient if said treatment threshhold was not met or to discontinue or suspend treatment of said patient if the treatment threshhold was met.
  • Also disclosed herein are methods of evaluating whether an immune response was induced in a patient by a treatment comprising the steps of: obtaining a first sample of a patient's blood; labelling the cells from said sample to detect the presence of cell surface markers CD27, CD28, and CD45RA; counting the number of cells in the sample that are positive for CD27, CD28, and CD45 RA; following administration to said patient of a treatment, obtaining a second sample of said patient's blood; labelling the cells from said second sample to detect the presence of cell surface markers CD27, CD28, and CD45RA; counting the number of cells in said second sample that are positive for CD27, CD28, and CD45 RA; and determining whether there has been an increase or decrease in the number or proportion of cells that are positive for CD27, CD28, and CD45RA in said first and second sample; wherein an increase indicates that an immune response was induced in the patient.
  • the treatment is the administration to the patient of autologous mature DCs that were produced in vitro.
  • Aslo disclosed herein is a method of stimulating immune effector cells, comprising culturing said cells in the presence of PME-CD40L DCs to produce stimulated immune effector cells.
  • the disclosure provides a method of enhancing immunity in a subject comprising administering to the subject an effective amount of such stimulated immune effector cells (T SCM cells).
  • T SCM cells stimulated immune effector cells
  • Introducing or administering immune cells into a subject is generally referred to as adoptive transfer therapy and is intended to help stimulate the subject's immune response.
  • adoptive transfer therapy is known in the art and has been demonstrated in a number of studies, such as, for example, Cobbold et al. (2005) J. Exp. Med. 3: 379-86 and Schmitt et al. (2011) Transfusion 3: 591-99 .
  • compositions described herein are useful to raise an immune response in a subject by administering to the subject an effective amount of the enriched population of cells, e.g., T SCM cells.
  • the cells can be allogeneic (heterologous) or autologous to the subject. They can be administered to a subject to raise or induce an immune response in a subject in a method comprising administering to the subject an effective amount of the enriched populations as described above.
  • the educated effector cells can also be used to enhance immunity in a subject by delivering to the subject an effective amount of these cells.
  • T SCM cells can be administered to a patient infected with HIV to increase the immune response and/or to help inhibit opportunistic infection(s), or T SCM cells can be administered to a cancer patient to increase the immune response to the cancerous cells.
  • an effective amount of T SCM cells is one that increases any measure of immune response by a statistically significant amount.
  • the disclosure also provides in vitro methods involving T SCM cells.
  • dendritic cells loaded with an antigen can be used to produce and expand T SCM cells in vitro.
  • T SCM cells educated in vitro e.g., by coculturing with DCs presenting antigen
  • target cells are typically cancer cells, or infected cells which express unique antigenic peptides on their MHC class I surfaces; thus, for example, target cells can be cells infected with HIV or can be cancer cells.
  • T SCM cells produced by the methods of this invention can be administered directly to the subject (e.g., a human patient) to produce T cells active against a selected immunogen.
  • the cells are administered in any suitable manner, for example, with pharmaceutically acceptable carriers, which are well known in the art.
  • the cells can be provided in a composition also comprising a pharmaceutically acceptable carrier.
  • a medicament comprising T SCM cells for use in treating a patient.
  • the T SCM cells disclosed herein are suitable for use in preparing a medicament for treating a patient, such as a cancer patient or a patient infected with HIV.
  • Suitable methods of administering cells in the context of the present disclosure to a subject are available, and, although more than one route can be used to administer a particular cell compositon, a particular route can often provide a more immediate and effective reaction than another route.
  • Administration can be by methods known in the art to successfully deliver a cell into ultimate contact with a subject's blood or tissue cells.
  • Preferred routes of administration include but are not limited to intradermal and intravenous administration.
  • compositions of the present disclosure are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure. Most typically, quality controls (microbiology, clonogenic assays, viability tests), are performed and the cells are infused into the patient from whom they were derived and/or isolated, preceded by the administration of diphenhydramine and hydrocortisone. See, for example, Korbling et al. (1986) Blood 67:529-532 and Haas et al. (1990) Exp. Hematol. 18:94-98 .
  • the dose of cells (e.g., T SCM cells) administered to a subject is in an amount effective to achieve the desired beneficial therapeutic response in the subject over time, or to inhibit growth of cancer cells, or to inhibit infection (i.e., an "effective amount"); those of skill in the art recognize however that the patient can benefit from an increase in any measure of the immune response, even if a complete cure is not achieved.
  • a method of adoptive transfer therapy of the disclosure can be practiced by obtaining and saving blood samples from the patient prior to infusion for subsequent analysis and comparison.
  • at least about 10 4 to 10 6 and typically between 1 x 10 8 and 1 x 10 10 cells can be infused ( e.g., intravenously or intraperitoneally) into a 70 kg patient over roughly 60-120 minutes.
  • Vital signs and oxygen saturation by pulse oximetry can be closely monitored, and blood samples obtained at intervals following infusion (e.g., 5 minutes and 1 hour) and saved for analysis.
  • Cell re-infusions can be repeated roughly every month for a total of 10-12 treatments in a one year period, if deemed appropriate.
  • infusions can be performed on an outpatient basis at the discretion of the clinician. If the re-infusion is given as an outpatient treatment, the participant is monitored for at least 4 hours following the treatment.
  • cells of the present disclosure can be administered at a rate determined by the effective dose, the LD-50 of the cell type (or other measure of toxicity), and the side-effects of the cell type at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.
  • the cells of this disclosure can supplement other treatments for a condition by known conventional therapy, including cytotoxic agents, nucleotide analogues and biologic response modifiers.
  • biological response modifiers are optionally added for treatment; for example, the cells are optionally administered with an adjuvant, or cytokine such as GM-CSF, IL-12 or IL-2.
  • the IFN- ⁇ R agonist used in the PME-CD40L process can be IFN- ⁇ or a biologically active fragment thereof.
  • the IFN- ⁇ is a mammalian IFN- ⁇ , most preferably a human IFN- ⁇ .
  • the cDNA and amino acid sequence of human IFN- ⁇ are shown in SEQ ID NOs: 5 and 6 of WO2007117682 , respectively.
  • the IFN- ⁇ has the sequence shown in SEQ ID NO:6, or a fragment thereof.
  • the IFN-yR comprises a polypeptide having at least 80% sequence identity with SEQ ID NO:6 of WO2007117682 .
  • the IFN- ⁇ R agonist has at least 85%, 90%, 95%, 97%, 98% or 99% sequence identity with SEQ ID NO:6 of WO2007117682 .
  • Methods for testing the activity of IFN- ⁇ R agonists are known in the art (see, for example, Magro et al. (2004) Br. J. PharmacoL 142: 1281-92 ).
  • Immature DCs can be signaled by adding an IFN- ⁇ R agonist the culture medium, or by expressing the IFN- ⁇ R agonist in the dendritic cell.
  • the DC is transfected with an mRNA encoding an IFN- ⁇ R agonist, such as SEQ ID NO:6 of WO2007117682 , or a biologically active fragment thereof. Signaling would then occur upon translation of the mRNA within the dendritic cell.
  • the IFN- ⁇ R agonist is added to the culture medium containing immature DCs.
  • the culture medium further comprises PGE 2 and/or GM-CSF plus IL-4 or IL-13.
  • the second signal used to produce PME-CD40L DCs is a transient signal with a CD40 agonist.
  • the signal can be considered transient if the DCs are loaded with an mRNA encoding a CD40 agonist, or if medium containing a CD40 agonist is removed from the DCs.
  • persistent expression of a CD40 agonist polypeptide such as constitutive expression of CD40L from a lentiviral vector, is not considered transient expression.
  • the CD40 agonist signal can also be considered transient if the DCs are loaded/transfected with RNA or with an expression vector encoding a CD40 agonist, provided that either: 1) the promoter driving CD40 agonist expression is not constitutive in DCs, or 2) the expression vector does not integrate into the DC genome or otherwise replicate in DCs.
  • the CD40 agonist is a CD40L polypeptide or a CD40 agonistic antibody.
  • ligands that bind CD40 may act as a CD40 agonist, for example, a CD40 agonist can be an aptamer that binds CD40.
  • the CD40 agonist is delivered as mRNA encoding CD40L.
  • Administration of the second signal comprising CD40L to the cells by transfection of immature or mature DCs with CD40L mRNA produces the modified PME-CD40L DCs that induce immunostimulatory responses rather than immunosuppressive ones.
  • CD40L-mRNA-transfected dendritic cells are cultured in medium containing IFN- ⁇ (and optionally PGE 2 ) immediately after transfection and thus prior to translation of the CD40L mRNA to produce an effective amount of a CD40L signal.
  • IFN- ⁇ is added after transfection with CD40L mRNA
  • the dendritic cells receive the IFN- ⁇ signal prior to the signal that results from the translation of the CD40L mRNA.
  • the order in which the agents are delivered to the cells is important only in that CD40L signaling must occur after IFN- ⁇ signaling.
  • the signaling of the DCs can occur in vivo or ex vivo, or alternatively one or more signaling step may occur ex vivo and the remaining steps of the method can occur in vivo.
  • CD40 Ligand encompasses any polypeptide or protein that specifically recognizes and activates the CD40 receptor and activates its biological activity.
  • the term includes transmembrane and soluble forms of CD40L.
  • the CD40 agonist is a mammalian CD40L, preferably a human CD40L.
  • a human CD40L cDNA and the corresponding amino acid sequence are shown in SEQ ID NOs:1 and 2 of WO2007117682 , respectively.
  • the method comprises the sequential steps of: (a) signaling isolated immature dendritic cells (iDCs) with a first signal comprising an interferon gamma receptor (IFN- ⁇ R) agonist and a TNF- ⁇ R agonist, to produce IFN-yR-agonist-signaled dendritic cells; and (b) signaling said IFN- ⁇ R-agonist-signaled dendritic cells with a second transient signal comprising an effective amount of a CD40L polypeptide to produce CD83 + CCR7 + mature dendritic cells, wherein the CD40L polypeptide consists essentially of amino acid residues 21-261 of SEQ ID NO:2 of WO2007117682 or a polypeptide having at least 80% sequence identity to amino acid residues 21-261 of SEQ ID NO:2 of WO2007117682 .
  • the method comprises the sequential steps of: (a) culturing isolated immature dendritic cells (iDCs) with an interferon gamma receptor (IFN- ⁇ R) agonist in the presence of a TNF- ⁇ R agonist and PGE 2 for approximately 12 to 30 hours to produce CD83 + mature dendritic cells; and (b) approximately 12 to 30 hours after initiating step (a), transfecting said CD83 + mature dendritic cells (mDCs) with mRNA encoding a CD40L polypeptide consisting of amino acid residues 21-261 of SEQ ID NO:2 of WO2007117682 and an mRNA encoding one or more antigens to produce CD83 + CCR7 + mature dendritic cells.
  • iDCs isolated immature dendritic cells
  • IFN- ⁇ R interferon gamma receptor
  • CD40 Ligand was cloned in 1993 and reported by Gauchat et al. (1993) FEBS Lett. 315: 259 . Shorter soluble forms of the cell-associated full-length 39 kDa form of CD40 Ligand have been described with molecular weights of 33 kDa and 18 kDa ( Graf et al. (1995) Eur. J. Immunol. 25: 1749 ; Ludewig et al. (1996) Eur. J. Immunol. 26: 3137 ; Wykes et al. (1998) Eur. J. Immunol. 28: 548 ).
  • the 18 kDa soluble form generated via intracellular proteolytic cleavage lacks the cytoplasmic tail, the transmembrane region, and parts of the extracellular domain, but conserves the CD40 binding domain and retains the ability to bind to CD40 receptor; therefore, it is an example of a CD40-receptor-signaling agent. See Graf et al. (1995) supra. U.S. Patent Nos. 5,981,724 and 5,962,406 also disclose DNA sequences encoding human CD40 Ligand (CD40L), including soluble forms of CD40L.
  • CD40L human CD40 Ligand
  • the open reading frame for CD40L is represented by nucleotides 40 to 822 of SEQ ID NO.1 of WO2007117682 , while the TGA stop codon is at position 823 to 825.
  • a sequence containing a silent mutation may be used; for example, a variant due to codon degeneracy of the 102 nd codon in the CD40L sequence (nucleotides 343 to 345 of SEQ ID NO:1 of WO2007117682 ) changes the "AAA" codon to an "AAG” codon, both of which code for Lys.
  • CD40L also useful in methods to produce mature DCs are truncated CD40L (residues 47 to 261 of SEQ ID NO:2 of WO2007117682 , encoded by nucleotide residues 178 to 825 of SEQ ID NO:1 of WO2007117682 ) and CD40L fragments encoded by nucleotides 43 to 825 of said SEQ ID NO:1, 181 to 825 of said SEQ ID NO:1, 193 to 825 of said SEQ ID NO:1, 376 to 825 of said SEQ ID NO:1, 379 to 825 of said SEQ ID NO:1 and 400 to 825 of said SEQ ID NO:1 of WO2007117682 .
  • the CD40L polypeptide is a polypeptide comprising the sequence set forth in SEQ ID NO:2 of WO2007117682 .
  • any polypeptide fragment of the full-length CD40L may be used in the methods if the polypeptide acts as a CD40 ligand by specifically binding CD40 and producing biological activity.
  • the CD40L polypeptide is encoded by an mRNA comprising a polynucleotide encoding CD40L and further comprising a 3' untranslated sequence known in the art such as the CD40 receptor 3' UTR, the untranslated region of the final exon of the human beta-actin 3' UTR, the minimal functional element of the human beta-actin 3' UTR, and the simian rotavirus gene 6 3' UTR.
  • the mRNA may also, or alternatively, comprise a 5' untranslated sequence known in the art such as the human Hsp70 5' UTR, the mouse VEGF 5' UTR, the minimal functional element of the mouse VEGF 5' UTR, the spleen necrosis virus LTR RU5 region, and the tobacco etch virus 5' leader sequence.
  • these RNAs are capped and polyadenylated.
  • the CD40 receptor can also be activated by use of CD40 agonist antibodies, antibody fragments, derivatives and variants thereof, which are known in the art.
  • CD40 agonist antibodies can be purchased from commercial vendors such as Mabtech (Nacka, Sweden).
  • the literature also provides examples otf CD40 agonist antibodies and antibody fragments. See, e.g., Osada et al. (2002) 25(2): 176 and Ledbetter et al. (1997) Crit. Reviews in Immunol. 17: 427 .
  • Modified CD40L can also be used in methods of dendritic cell maturation; for example, CD40L includes polypeptides that have been altered through addition, subtraction, or substitution, either conservatively or non-conservatively, of any number of amino acids, provided that the resulting protein binds CD40 on the surface of DCs.
  • Steps of the methods described herein can be practiced in vivo or ex vivo, as appropriate.
  • the method can be practiced in an open or closed system.
  • Methods and systems for culturing and enriching cell populations are known in the art. See Examples 1 and 2 of U.S. Patent Publication No. 2004/0072347 ; see also U.S. Patent Publication No. 2003/0235908 , which describes closed systems for cell expansion.
  • the methods of preparing mature DCs can be further modified by contacting the cell with an effective amount of a cytokine or co-stimulatory molecule, e.g., GM-CSF, IL-4 and PGE 2 .
  • a cytokine or co-stimulatory molecule e.g., GM-CSF, IL-4 and PGE 2 .
  • effective amounts of IL-1 ⁇ and/or IL-6 are specifically excluded from the culture.
  • the method used to produce PME-CD40L DCs can also include delivering to the immature or mature DCs an effective amount of an antigen which will be then be processed and presented by the mature DCs.
  • Antigens can be naturally occurring or recombinantly produced.
  • the antigens can be delivered to the cells as polypeptides or proteins or as nucleic acids encoding them using methods known in the art.
  • one or more polynucleotides encoding one or more antigens are introduced into the iDCs, signaled DCs or CCR7 + mature DCs by methods known to those of skill in the art such as electroporation.
  • the polynucleotide is an mRNA.
  • the antigen or antigen-encoding mRNA is introduced together with an mRNA encoding a CD40 agonist or substantially concurrent with CD40 agonist signaling.
  • an mRNA encoding the antigen is introduced into the DC, and may be cotransfected with an mRNA encoding a CD40L polypeptide.
  • the dendritic cells are cultured in medium containing the antigen.
  • the DCs then take up and process the antigen on the cell surface in association with MHC molecules.
  • the DCs are loaded with antigen by transfection with a nucleic acid encoding the antigen, for example, an mRNA.
  • Methods of transfecting DCs are known to those of skill in the art.
  • An antigen can be a single known antigen or can be a collection of antigens.
  • a collection of antigens may come from one particular source, such as for example a patient's cancer cells or HIV-infected cells, or may come from several sources, such as for example HIV-infected cells from several different patients.
  • Antigens for use in methods of producing PME-CD40L DCs include, but are not limited to, antigens from: pathogens, pathogen lysates, pathogen extracts, pathogen polypeptides, viral particles, bacteria, proteins, polypeptides, cancer cells, cancer cell lysates, cancer cell extracts, and cancer-cell-specific polypeptides.
  • antigens that can be used to produce PME-CD40L DCs include such well-known antigens as MART-1; the resulting PME-CD40L DCs presenting MART-1 can then be used to expand populations of MART-1-reactive T SCM CTLs (see, e.g., data shown in Figure 1 and discussed in Example 2).
  • the methods described herein further comprise introducing into iDCs, signaled DCs or CCR7 + mature DCs one or more antigens or a polynucleotide(s) encoding one or more antigens to produce an antigen-loaded CCR7 + mature DCs.
  • the antigen or antigen-encoding polynucleotide e.g., mRNA
  • mRNA encoding one or more antigens can be transfected into cells at the same time as other mRNA, such as mRNA encoding CD40L, or at a different time.
  • the antigen may be delivered in its "natural" form in that no human intervention was involved in preparing the antigen or inducing it to enter the environment in which it encounters the DC.
  • the antigen may comprise a crude preparation, for example of the type that is commonly administered in a conventional allergy shot or the antigen may comprise a tumor lysate.
  • the antigen may alternatively be substantially purified, e.g., at least about 90% purified or isolated.
  • An antigen that is a peptide may be generated, for example, by proteolytic cleavage of isolated proteins using methods known in the art, or may be chemically synthesized, e.g., on a commercially-available, automated synthesizer. Also, recombinant techniques may be employed to create a nucleic acid encoding the peptide of interest, and to express that peptide under desired conditions.
  • the antigen can alternatively have a structure that is distinct from any naturally-occurring compound.
  • the antigen is a "modified antigen" having a structure that is substantially identical to that of a naturally-occurring antigen but that includes one or more deviations from the exact structure of the naturally-occurring compound.
  • a modified antigen as compared with that protein or polypeptide antigen may have an amino acid sequence that differs from that of the naturally-occurring antigen in the addition, substitution, or deletion of one or more amino acids, and/or might include one or more amino acids that differ from the corresponding amino acid in the naturally-occurring antigen by the addition, substitution, or deletion of one or more chemical moieties covalently linked to the amino acid.
  • the naturally-occurring and modified antigens share at least one region of at least 5 amino acids that are at least approximately 75% identical.
  • the antigens can also be modified by linking a portion of sequence from a first polypeptide (e.g., a first antigen) to a portion of sequence from a second polypeptide (e.g., a second antigen, a signal sequence, a transmembrane domain, a purification moiety, etc .) by means of a peptide bond.
  • a first polypeptide e.g., a first antigen
  • a second polypeptide e.g., a second antigen, a signal sequence, a transmembrane domain, a purification moiety, etc .
  • antigen to be employed in any particular composition or application will depend on the nature of the particular antigen and of the application for which it is being used, as will readily be appreciated by those of skill in the art, and can be adjusted by one of skill in the art to provide the necessary amount of expression.
  • the antigen may be generated, for example, by proteolytic cleavage of isolated proteins. Any of a variety of cleavage agents may be utilized including, but not limited to, pepsin, cyanogen bromide, trypsin, chymotrypsin, etc. Alternatively, peptides may be chemically synthesized, preferably on an automated synthesizer such as is available in the art (see, for example, Stewart et al., (1984) Solid Phase Peptide Synthesis, 2d. Ed., Pierce Chemical Co. ).
  • recombinant techniques may be employed to create a nucleic acid encoding the peptide of interest, and to express that peptide under desired conditions (e.g., in a host cell or an in vitro expression system from which it can readily be purified).
  • the antigen is from a cancer cell or a pathogen.
  • the cancer cell can be any type of cancer cell, including a renal cancer cell (e.g., from renal cell carcinoma), a multiple myeloma cell or a melanoma cell.
  • Preferred pathogens include HIV and HCV.
  • the antigen is delivered to the DCs in the form of RNA isolated or derived from a cancer cell or a pathogen or pathogen-infected cell (e.g., an HIV-infected cell).
  • Methods for RT-PCR of RNA extracted from any cell (e.g., a cancer cell or pathogen cell), and in vitro transcription are disclosed in WO2006031870 (Nicolette et al. ) and U.S. Pub. 20070248578 (Tcherepanova et al. ) .
  • both cytokine and antigen are to be delivered to an individual, they may be provided together or separately. When they are delivered as polypeptides or proteins, they can be delivered in a common encapsulation device or by means of physical association ( e.g., by covalent linkage). Similarly, the compounds can be provided together when polynucleotides encoding both are provided; for example, genes for both may be provided as part of the same nucleic acid molecule. In some examples, this nucleic acid molecule may be prepared so that both factors are expressed from a single contiguous polynucleotide, for example as a fusion protein in which the cytokine and the antigen are covalently linked to one another via a peptide bond.
  • the genes may be linked to the same or equivalent control sequences, so that both genes become expressed within the individual in response to the same stimuli.
  • control sequences active in different host cells under different conditions are known in the art. These control sequences, including constitutive control sequences, inducible control sequences, and repressible control sequences, can be used, though inducible or repressible sequences are often preferred for applications in which additional control over the timing of gene expression is desired.
  • T SCM cells of the disclosure are administered to a patient in autologous or allogeneic adoptive transfer therapy who is also being treated with an anti-PD-1 therapy, for example, an anti-PD-1 antibody.
  • Anti-PD-1 antibodies and the use thereof are known in the art, for example, as described in Hamid et al. (2013) New England J. Med. 369: 2 .
  • methods of treating a patient comprising the steps of administering T SCM cells and administering an anti-PD-1 antibody.
  • Dendritic cells can be transfected with nucleic acids (including RNA encoding antigens) by methods known in the art, which include but are not limited to calcium phosphate precipitation, microinjection, or electroporation.
  • the nucleic acids can be added alone or in combination with a suitable carrier, e.g., a pharmaceutically acceptable carrier such as phosphate buffered saline.
  • a suitable carrier e.g., a pharmaceutically acceptable carrier such as phosphate buffered saline.
  • the nucleic acid can be incorporated into an expression or insertion vector for incorporation into the cells.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are known in the art.
  • Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI).
  • Stratagene La Jolla, CA
  • Promega Biotech Promega Biotech
  • consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
  • vectors are viruses, such as baculovirus and retrovirus, bacteriophage, adenovirus, adeno-associated virus, cosmid, plasmid, fungal vectors and other vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. Any suitable vector or delivery method may be used and can readily be selected by one of skill in the art.
  • viruses such as baculovirus and retrovirus, bacteriophage, adenovirus, adeno-associated virus, cosmid, plasmid, fungal vectors and other vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. Any suitable vector or delivery method may be used and can readily be selected by one of skill in the art.
  • Polynucleotides are inserted into vectors using methods and reagents known in the art, including for example, restriction enzymes, synthetic nucleic acid linkers, and oligonucleotides; other functional sequences can be included in the vector and/or associated with the polynucleotide as needed, including, for example, selectable marker genes, enhancer sequences, promoter sequences, start and stop codons, transcription termination signals, RNA processing signals, origins of replication, multiple cloning sites, and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Other means and functional sequences are known and available in the art and can also be used.
  • RNA is delivered to the cell as mRNA.
  • RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell or by in vitro transcription. If a host cell is used, DNA can be inserted into the cell by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA; the RNA can then be isolated using methods well known to those of skill in the art, for example, as set forth in Sambrook, ed.
  • an appropriate gene delivery vehicle e.g., liposome, plasmid or vector
  • RNA can be isolated using various lytic enzymes or chemical solutions according to Sambrook (2001), supra, or extracted by nucleic-acid-binding resins or other commercially-available products following the instructions provided by the manufacturer.
  • the CD40L expression cassette contains a promoter suitable for in vitro transcription, such as the T7 promoter or SP6 promoter.
  • a promoter suitable for in vitro transcription such as the T7 promoter or SP6 promoter.
  • the in vitro transcribed CD40L or CD40 agonist mRNA is optimized for stability and efficiency of translation, such as, for example, as demonstrated by SEQ ID NO:13 of WO2007117682 , which represents an optimized CD40L mRNA wherein ATG codons in the 5' untranslated region have been altered to avoid incorrect initiation of translation.
  • mRNA stability and/or translational efficiency can also be increased by including 3' UTRs and or 5' UTRs in the mRNA.
  • 3' UTRs include those from human CD40, beta-actin and rotavirus gene 6.
  • 5' UTRs include those of CD40L and the translational enhancers in the 5' UTRs of Hsp70, VEGF, spleen necrosis virus RU5, and tobacco etch virus.
  • CD40L expression is normally regulated in part by 3' UTR-mediated mRNA instability, and therefore a large portion of the CD40L 3' UTR is not included in the current CD40L mRNA.
  • CD40L is not normally expressed in DCs, but the CD40 Receptor is expressed in DCs and its expression does not seem to be regulated post-transcriptionally, particularly at the level of mRNA stability, so including the CD40 Receptor 3' UTR or an active fragment thereof at the 3' end or region of the CD40L mRNA would give the RNA 3' untranslated sequence similar to naturally occurring CD40 messages without imparting any unwanted regulatory activity.
  • the benefits and disadvantages of other 5' and 3' UTRs are also known in the art, and one of skill in the art is able to select appropriate UTRs for use in a particular cell type or situation.
  • Polypeptides and proteins are needed to perform methods of DC maturation; many are commercially available, and others can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or 431A, Foster City, CA, USA.
  • the synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the proteins and polypeptides can be obtained by any known method, including any recombinant method.
  • Peptides that may be used in the methods of the disclosure include peptides comprising natural and/or unnatural or synthetic amino acids, including D- and L-amino acids, amino acid analogs, and peptidomimetics.
  • antibodies can be monoclonal or polyclonal. They can be antibody derivatives or antibody variants, such as, for example, linear antibodies. They can be chimeric, humanized, or totally human. Using a protein or a polypeptide one of skill in the art can generate additionally antibodies which specifically bind to the receptor. A functional fragment or derivative of an antibody also can be used including Fab, Fab', Fab2, Fab'2, and single chain variable regions. Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes, etc.
  • Antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen at least 2, 5, 7, and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific. Techniques for making such partially to fully human antibodies are known in the art and any such techniques can be used.
  • RNA levels such as IL-12 p35 mRNA or CD40L mRNA
  • assaying for an alteration in mRNA level such as IL-12 p35 mRNA or CD40L mRNA
  • the nucleic acid contained in a sample can be first extracted using methods known in the art; commercial kits are available.
  • the mRNA contained in the extracted nucleic acid sample can then be detected by hybridization (e.g., Northern blot analysis) and/or amplification procedures using nucleic acid probes and/or primers, respectively, according to standard procedures known to those of ordinary skill in the art.
  • hybridization e.g., Northern blot analysis
  • amplification procedures using nucleic acid probes and/or primers, respectively, according to standard procedures known to those of ordinary skill in the art.
  • Nucleic acid molecules having at least 10 nucleotides and exhibiting sequence complementarity or homology to the nucleic acid to be detected can be used as hybridization probes or primers in diagnostic methods. It is known in the art that a "perfectly matched" probe is not needed for a specific hybridization. Minor changes in probe sequence achieved by substitution, deletion or insertion of a small number of bases do not affect the hybridization specificity. In general, as much as 20% base-pair mismatch (when optimally aligned) can be tolerated.
  • a probe useful for detecting CD40L mRNA is at least about 80% identical to the homologous region of comparable size contained in a previously identified sequence, e.g., see SEQ ID NOS: 1 or 3 of WO2007117682 .
  • the probe is at least 85% or even at least 90% identical to the corresponding gene sequence after alignment of the homologous region.
  • the total size of the fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the particular nucleic acid segment. Smaller fragments of the gene will generally find use in hybridization examples, wherein the length of the complementary region may be varied, such as between about 10 and about 100 nucleotides, or even full length according to the complementary sequences of interest for detection.
  • Nucleotide probes having sequences complementary to a target sequence of a nucleotide over stretches greater than about 10 nucleotides in length will increase stability and selectivity of the hybrid between the probe and the target nucleotide, thereby improving the specificity of particular hybrid molecules obtained.
  • One of skill in the art can design nucleic acid molecules having complementary stretches of more than about 25 and even more preferably more than about 50 nucleotides in length, or even longer where desired.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology with two priming oligonucleotides (for example, as described in U.S. Patent No. 4,603,102 ) or by introducing selected sequences into recombinant vectors for recombinant production.
  • nucleic acid sequences of the present disclosure in combination with an appropriate means, such as a label, for detecting hybridization and therefore complementary sequences.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • a fluorescent label or an enzyme tag such as urease, alkaline phosphatase, or peroxidase can also be used.
  • enzyme tags colorimetric indicator substrates are knownin the art which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • hybridization reactions can be performed under conditions of different "stringency.” Relevant conditions include temperature, ionic strength, time of incubation, the presence of additional solutes in the reaction mixture such as formamide, and the washing procedure. Higher stringency conditions are those conditions such as higher temperature and lower sodium ion concentration, which require higher minimum complementarity between hybridizing elements for a stable hybridization complex to form. Conditions that increase the stringency of a hybridization reaction are widely known in the art.
  • One of skill in the art can also utilize, detect, and quantify the amount or level of expression of mRNA using quantitative PCR or high throughput analysis such as Serial Analysis of Gene Expression (SAGE) as described in Velculescu et al. (1995) Science 270:484-487 . Other techniques are also known in the art.
  • SAGE Serial Analysis of Gene Expression
  • PCR The Basics, Second Edition (Taylor & Francis Group, New York, NY )).
  • PCR conditions used for each application reaction are empirically determined. A number of parameters influence the success of a reaction, including annealing temperature and time, extension time, Mg 2+ ATP concentration, pH, and the relative concentration of primers, templates, and deoxyribonucleotides; adjustment of these parameters to achieve the desired result is known to those of skill in the art.
  • the resulting DNA fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
  • a specific amplification of differentially expressed genes of interest can be verified by demonstrating that the amplified DNA fragment has the predicted size, exhibits the predicated restriction digestion pattern, and/or hybridizes to the correct cloned DNA sequence.
  • Other methods for detecting gene expression are known to those skilled in the art. See, for example, International PCT Application No. WO 97/10365 , U.S. Pat. Nos. 5,405,783 , 5,412,087 and 5,445,934 , 5,405,783 ; 5,412,087 ; 5,445,934 ; 5,578,832 ; and 5,631,734 .
  • radioimmunoassays include, but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), "sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.
  • an effective amount of a cytokine and/or co-stimulatory molecule is delivered to the cells or patient, in vitro or in vivo.
  • agents can be delivered as polypeptides, proteins or alternatively, as the polynucleotides or genes encoding them.
  • Cytokines, co-stimulatory molecules and chemokines can be provided as impure preparations ( e.g., isolates of cells expressing a cytokine gene, either endogenous or exogenous to the cell) or in a "purified" form. Purified preparations are preferably at least about 90% pure, or alternatively, at least about 95% pure, or yet further, at least about 99% pure.
  • genes encoding the cytokines or inducing agents may be provided, so that gene expression results in cytokine or inducing agent production either in the individual being treated or in another expression system (e.g., an in vitro transcription/translation system or a host cell) from which expressed cytokine or inducing agent can be obtained for administration to the individual.
  • another expression system e.g., an in vitro transcription/translation system or a host cell
  • the immunogenicity of the T cells produced by the methods described herein can be determined by well known methodologies, including but not limited to the following:
  • a cell includes a plurality of cells, including mixtures thereof.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of' when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Polypeptides or protein that "consist essentially of' a given amino acid sequence are defined herein to contain no more than three, preferably no more than two, and most preferably no more than one additional amino acids at the amino and/or carboxy terminus of the protein or polypeptide.
  • Nucleic acids or polynucleotides that "consist essentially of' a given nucleic acid sequence are defined herein to contain no more than ten, preferably no more than six, more preferably no more than three, and most preferably no more than one additional nucleotide at the 5' or 3' terminus of the nucleic acid sequence.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure. Examples defined by each of these transition terms are within the scope of this disclosure.
  • antigen is well understood in the art and includes any substance which is immunogenic, i.e., an immunogen. It will be appreciated that the use of any antigen is envisioned for use in the present disclosure and thus includes but is not limited to a self-antigen (whether normal or disease-related), an antigen of an infectious agent (e.g., a microbial antigen, viral antigen, etc .), or some other foreign antigen (e.g., a food component, pollen, etc .).
  • infectious agent e.g., a microbial antigen, viral antigen, etc .
  • foreign antigen e.g., a food component, pollen, etc .
  • antigen or “immunogen” applies to collections of more than one immunogen, so that immune responses to multiple immunogens may be modulated simultaneously.
  • the term includes any of a variety of different formulations of immunogen or antigen.
  • a “native” or “natural” or “wild-type” antigen is a polypeptide, protein or a fragment thereof which contains an epitope, which has been isolated from a natural biological source, and which can specifically bind to an antigen receptor when presented in a subject as an MHC/peptide complex, in particular a T cell antigen receptor (TCR).
  • TCR T cell antigen receptor
  • tumor associated antigen refers to an antigen that is associated with a tumor.
  • TAAs include gp100, MART and MAGE.
  • Other tumor antigens may be specific to a particular tumor in a particular patient.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • the proteins encoded by the MHC are known as “MHC molecules” and are classified into Class I and Class II MHC molecules.
  • Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8 + T cells.
  • Class I molecules include HLA-A, B, and C in humans.
  • Class II MHC molecules are known to function in CD4 + T cells and, in humans, include HLA-DP, -DQ, and -DR.
  • APCs antigen presenting cells
  • APCs can be intact whole cells such as macrophages, B-cells, endothelial cells, activated T-cells, and dendritic cells; or other molecules, naturally occurring or synthetic, such as purified MHC Class I molecules complexed to ⁇ 2-microglobulin.
  • CTL cytotoxic T-lymphocyte
  • DCs dendritic cells
  • APCs a variety of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues
  • dendritic cells constitute the most potent and preferred APCs in the organism.
  • DCs can be differentiated from monocytes but are phenotypically distinct from monocytes; for example, CD 14 antigen is not found in dendritic cells but is possessed by monocytes.
  • mature dendritic cells are not phagocytic, whereas monocytes are strongly phagocytosing cells. It has been shown that mature DCs can provide all the signals necessary for T cell activation and proliferation.
  • immune effector cells refers to cells capable of binding an antigen and which mediate an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.
  • T cells T cells
  • B cells monocytes
  • macrophages NK cells
  • CTLs cytotoxic T lymphocytes
  • CTLs cytotoxic T lymphocytes
  • a "naive" immune effector cell is an immune effector cell that has never been exposed to an antigen capable of activating that cell. Activation of naive immune effector cells requires both recognition of the peptide:MHC complex and the simultaneous delivery of a costimulatory signal by a professional APC in order to proliferate and differentiate into antigen-specific armed effector T cells.
  • the term "educated, antigen-specific immune effector cell” is an immune effector cell as defined above which has previously encountered an antigen. In contrast to its naive counterpart, activation of an educated, antigen-specific immune effector cell does not require a costimulatory signal; recognition of the peptide:MHC complex is sufficient.
  • Immuno response broadly refers to the antigen-specific responses of lymphocytes to foreign substances. Any substance that can elicit an immune response is said to be “immunogenic” and is referred to as an "immunogen".
  • An immune response of this disclosure can be humoral (via antibody activity) or cell-mediated (via T cell activation).
  • Activated when used in reference to a T cell, implies that the cell is no longer in Go phase, and begins to produce one or more of cytotoxins, cytokines and other related membrane-associated proteins characteristic of the cell type (e.g., CD8 + or CD4 + ), and is capable of recognizing and binding any target cell that displays the particular peptide/MHC complex on its surface, and releasing its effector molecules.
  • the phrase "inducing an immune response in a subject” or to induce an immune response in a subject is understood in the art and refers to an increase of at least about 2-fold, or alternatively at least about 5-fold, or alternatively at least about 10-fold, or alternatively at least about 100-fold, or alternatively at least about 500-fold, or alternatively at least about 1000-fold or more in an immune response to an antigen which can be detected or measured, after introducing the antigen into the subject, relative to the immune response (if any) before introduction of the antigen into the subject.
  • a treatment is considered to have induced an immune response to an antigen in a subject if an immune response is increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to the immune response exhibited by the subject to the antigen before the treatment.
  • An immune response to an antigen includes but is not limited to the production of an antigen-specific antibody or an increase in the production of antigen-specific antibodies; an increase or decrease in the amount or frequency of an identifiable immune cell type; and the production of an immune cell expressing on its surface a molecule which specifically binds to an antigen.
  • antigen-specific antibody can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, wherein, for example, binding of an antibody in a sample to an immobilized antigen is detected with a detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).
  • ELISA e.g., enzyme-labeled mouse anti-human Ig antibody
  • cytokine refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
  • Nonlimiting examples of cytokines which may be used alone or in combination in the practice of the present disclosure include interleukin-2 (IL-2), stem cell factor (SCF), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 ⁇ ), interleukin-1L (IL-1L), MIP-11, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO), IL-15, and 1L-17.
  • IL-2 interleukin-2
  • SCF stem cell factor
  • IL-3 interleukin-6
  • IL-12 interleukin-12
  • G-CSF granulocyte macrophage-colony stimulating factor
  • One example of the present disclosure includes culture conditions in which an effective amount of IL-1 ⁇ and/or IL-6 is excluded from the medium.
  • Cytokines are readily commercially available, and may be 'natural' purified cytokines or may be recombinantly produced.
  • polynucleotide “nucleic acid,” and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
  • polynucleotide includes, for example, a gene or gene fragment, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid molecule of the present disclosure may also comprise modified nucleic acid molecules.
  • mRNA refers to an RNA that can be translated in a dendritic cell. Such mRNAs typically are capped and have a ribosome binding site (Kozak sequence) and a translational initiation codon.
  • an RNA corresponding to a cDNA sequence refers to an RNA sequence having the same sequence as the cDNA sequence, except that the nucleotides are ribonucleotides instead of deoxyribonucleotides, as thymine (T) base in DNA is replaced by uracil (U) base in RNA.
  • peptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits may be linked by peptide bonds.
  • the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is relatively short, whereas if the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • a “conservative alteration" to a polypeptide or protein is one that results in an alternative amino acid of similar charge density, hydrophilicity or hydrophobicity, size, and/or configuration (e.g., Val for Ile).
  • a “nonconservative alteration” is one that results in an alternative amino acid of differing charge density, hydrophilicity or hydrophobicity, size and/or configuration (e.g., Val for Phe).
  • the means of making such modifications are well-known in the art.
  • genetically modified means containing and/or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny. In other words, it refers to any addition, deletion or disruption to a cell's endogenous nucleotides.
  • expression refers to the processes by which polynucleotides are transcribed into mRNA and mRNA is translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA of an appropriate eukaryotic host, expression may include splicing of the mRNA. Regulatory elements required for expression are known in the art and include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. Appropriate vectors for bacterial and/or eukaryotic expression are known in the art and are available commercially.
  • Under transcriptional control is a term understood in the art and indicates that transcription of a polynucleotide sequence (usually a DNA sequence) depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. "Operatively linked” refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • Examples of gene delivery vehicles include liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • Gene delivery is terms referring to the introduction of an exogenous polynucleotide into a host cell, regardless of the method used for the introduction.
  • Transfection refers to delivery of any nucleic acid to the interior of a cell and may include a variety of techniques such as: electroporation; protein-based, lipid-based and cationic-ion-based nucleic acid delivery complexes; viral vectors; "gene gun” delivery; and various other techniques known in the art.
  • the introduced polynucleotide can be stably maintained in the host cell or may be transiently expressed. In preferred examples, an mRNA is introduced into a DC and is transiently expressed.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are capable of mediating transfer of genes to mammalian cells and are known in the art.
  • sequence of a polynucleotide or portion thereof has a certain percentage of "sequence identity" to another sequence (for example, 80%, 85%, 90%, or 95%) when that percentage of bases or amino acids are the same when the two sequences are aligned and compared.
  • sequence identity for example, 80%, 85%, 90%, or 956% when that percentage of bases or amino acids are the same when the two sequences are aligned and compared.
  • the proper alignment and the percent sequence identity between two sequences can be determined using one of the well-known and publicly available BLAST alignment programs with default parameters.
  • isolated means separated from constituents, cellular and otherwise, with which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature.
  • an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome.
  • a mammalian cell, such as dendritic cell is isolated from an organism if it is removed from the anatomical site from which it is found in an organism.
  • a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than “concentrated” or less than “separated” than that of its naturally occurring counterpart.
  • “Host cell,” “target cell” or “recipient cell” are intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or proteins. It also is intended to include progeny of a single cell. In some instances, a progeny cell may not be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • the cells may be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.
  • a “subject” or “patient” is a mammal; in many examples, a patient is a human patient.
  • a subject or patient can also be any other mammal, including a monkey or ape, or any domestic animal such as a dog, cat, horse, etc.
  • cancer is meant the abnormal presence of cells which exhibit relatively autonomous growth, so that a cancer cell exhibits an aberrant growth phenotype characterized by a significant loss of cell proliferation control (i.e., it is neoplastic).
  • Cancerous cells can be benign or malignant. In various examples, cancer affects cells of the bladder, blood, brain, breast, colon, digestive tract, lung, ovaries, pancreas, prostate gland, or skin.
  • the definition of a cancer cell includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor, including metastasized cancer cells, in vitro cultures, and cell lines derived from cancer cells.
  • Cancer includes, but is not limited to, solid tumors, liquid tumors, hematologic malignancies, renal cell cancer, melanoma, breast cancer, prostate cancer, testicular cancer, bladder cancer, ovarian cancer, cervical cancer, stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, neuroblastoma, glioblastoma, retinoblastoma, leukemias, myelomas, lymphomas, hepatoma, adenomas, sarcomas, carcinomas, blastomas, etc.
  • a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition.
  • culturing refers to the in vitro maintenance, differentiation, and/or propagation of cells or in suitable media.
  • enriched is meant a composition comprising cells present in a greater percentage of total cells than is found in another composition, such as, for example, the tissues where they are present in an organism or a group or mixture of cells in which they were previously present.
  • T SCM cells are present in a higher percentage of total cells as compared to their percentage in an in vitro culture in which they were produced or cultured.
  • T SCM cells that are in an 'enriched' composition are present as more than 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the cells in that composition.
  • purified or isolated is intended that a cell type (e.g., T SCM cells) are present as more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or 99% of the cells in that composition.
  • depleted is intended that the frequency of that cell type is decreased in a particular composition or group of cells, e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more, or 100%.
  • enriching or “enrichment” as used herein is intended that cells are “enriched” using positive selection to selectively or preferentially remove them from a population or group of cells, or that cells are “enriched” using negative selection to selectively or preferentially remove other cells from a starting population or group of cells so that the desired cell type(s) remain behind.
  • Positive and/or negative selection can be readily accomplished using materials and techniques known in the art. For example, cells expressing a particular cell surface marker can be separated from other cells using monoclonal antibodies that bind to the marker and are coupled to columns or magnetic beads; the separation is readily performed according to standard techniques and/or manufacturer or provider directions.
  • cell surface marker (sometimes herein referred to as “marker” or “cell marker”) is intended a molecule expressed on the surface of a cell that can be detected, for example, using labeled antibodies or other means known in the art.
  • a cell surface marker can comprise a protein, glycoprotein, or group of proteins and/or glycoproteins.
  • a cell surface marker is known to correlate with or be indicative of a particular cell type or one or more cell functions. Certain cell populations can be identified by expression of a particular set or combination of markers, or some subset thereof.
  • positive expression or “positive for” with reference to a cell surface marker as used herein is generally intended that the marker is expressed at detectable levels on a cell or in a group of cells or population of cells.
  • “positive expression” or “positive for” is used to refer to cells that express a particular cell surface marker at levels significantly above background levels or “negative” levels, which can be evaluated by comparison to other cells or other groups or populations of cells, or can be a selected level of expression identified as background or negative.
  • One of skill in the art is familiar with techniques for detecting expression of a marker and for determining the level or levels of expression that distinguish "positive" expression from background or negative expression.
  • Cells that have "positive expression” or are “positive for” a particular marker can exhibit expression that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 100%, 150%, 200%, 300%, 400%, or 500% higher than background expression, or at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, or 100-fold or higher than background or "negative” expression.
  • the marker is expressed intracellularly but the expression is detectable using techniques known in the art.
  • a marker is detected with moieties that bind the marker (e.g., antibodies) that are coupled to a fluorescent label or other label that can be measured using a FACS device according to the manufacturers or provider's directions, for example, as demonstrated by the experiments described in the working examples herein.
  • moieties that bind the marker e.g., antibodies
  • a fluorescent label or other label that can be measured using a FACS device according to the manufacturers or provider's directions, for example, as demonstrated by the experiments described in the working examples herein.
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro or in vivo.
  • pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, Remington's Pharmaceutical Sciences, 18th Ed. (Mack Publ. Co., Easton (1990 )).
  • an "effective amount” is an amount sufficient to produce any beneficial or desired results, such as enhanced immune response, treatment, prevention or amelioration of a medical condition (disease, infection, etc ).
  • An effective amount can be administered in one or more administrations, applications or dosages. Suitable dosages will vary depending on body weight, age, health, disease or condition to be treated and route of administration; methods of determining an effective amount are known in the art.
  • an "effective amount" of AGS-004 treatments is the number of AGS-004 doses needed for a particular patient to exhibit a desired result such as a decrease in HIV viral load following ARTI (also referred to as ATI or STI), or to increase the frequency of T SCM s in the patient's blood (for example, as described elsewhere herein), or to exhibit a delayed time to HIV viral rebound following ARTI, or to meet criteria to remain without resuming ART following ARTI.
  • ARTI also referred to as ATI or STI
  • T SCM s in the patient's blood for example, as described elsewhere herein
  • These criteria can vary, but in some instances a patient must be restarted on ART if there are two consecutive tests showing that the patient's CD4+ T cell cound is below 350 cells/mL.
  • a patient in addition to the CD4+ T cell count criteria, a patient must also meet viral load criteria of less than 10,000 copies/mL or be restarted on ART. It is understood by those of skill in the art that any positive immune response can provide a benefit to a patient (e.g., an HIV patient or a cancer patient), even if the patient is not completely cured of the HIV infection or cancer, for example, by strengthening the patient's immune response so that other treatments may be more effective than they would have been otherwise.
  • a patient e.g., an HIV patient or a cancer patient
  • signaling means contacting an immature or mature dendritic cell with an IFN- ⁇ receptor agonist, a TNF- ⁇ receptor agonist, a CD40L polypeptide or other CD40 agonist.
  • such agonists are provided externally, ( e.g., in the cell culture medium).
  • the polypeptide agonist is provided via transfection of an immature or mature dendritic cell with a nucleic acid encoding the polypeptide.
  • a nucleic acid aptamer agonist could be provided in the medium or by transfection.
  • the term "mature dendritic cells” means dendritic cells that demonstrate elevated cell surface expression of co-stimulator molecule CD83, compared to immature DCs (iDCs).
  • PME-CD40L DCs were prepared essentially as described in Calderhead et al. ((2008) J. Immunother. 31: 731-41 ). Briefly, CD40L was cloned from activated T cells that had been stimulated with phorbol 12-myristate 13-acetate (PMA); RT-PCR was performed on total RNA from the T cells using gene-specific CD40L primers to amplify and clone CD40L.
  • Human PBMCs were isolated from leukapheresis collections from healthy volunteers by Ficoll-histopaque density centrifugation.
  • PBMCs were resuspended in culture medium and allowed to adhere to plastic flasks; nonadherent cells were removed and remaining cells were cultured in medium supplemented with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) for 5-6 days at 37°C, 5% CO 2 .
  • DCs were harvested, washed in PBS, re-suspended in chilled Viaspan® media (DuPont Pharma®), and placed on ice. DCs were mixed with CD40L mRNA and antigen-encoding mRNA and electroporated. Immediately after electroporation, DCs were washed and re-suspended in medium that was supplemented with GM-CSF and IL-4. DCs were cultured for either 4 or 24 hours at 37°C in low-adherence plates with additional maturation stimuli as described below.
  • Immature DCs were phenotypically matured on day 5 of culture by adding TNF- ⁇ , IFN- ⁇ , and PGE 2 .
  • DCs were harvested and electroporated with antigen and CD40L mRNA as described above, and cultured in media containing GM-CSF and IL-4 for 4 hrs prior to harvest or formulation for vaccine production.
  • DCs were harvested and re-suspended in chilled PBS/ 1% FCS, then mixed with phycoerythrin (PE) or FITC-conjugated antibodies specific for CD1a, CD209, human leukocyte antigen (HLA)-ABC, HLA-DR, CD80, CD86, CD38, CD40, CD25, CD123, CD83, CCR6, CCR7, CD70, and CD14; isotype-matched antibodies were used as controls.
  • fluorescence analysis was performed with a FACScalibur flow cytometer (BD BiosciencesTM) and CellQuest software (BD BiosciencesTM). Chemotaxis of DCs was measured by migration through a 8- ⁇ m pore size polycarbonate filter. IL-10 and IL-12 in the DC supernatants were determined using ELISA.
  • mature DCs electroporated with mRNAs were co-cultured with CD8+ purified T-cells.
  • the cells were cultured in media supplemented with IL-2 and IL-7; on day 4, the media was supplemented with IL-2.
  • the CD8 + cells were harvested and restimulated with DC in media supplemented with IL-2 and IL-7.
  • CTL assays were performed 3 days after the second or third stimulation.
  • PME-CD40L DCs expressing the MART-1 tumor antigen were used to expand a population of T SCM CTLs in vitro.
  • PME-CD40L DCs were produced and transfected with mRNA encoding the MART-1 antigen and cocultured with PBMCs isolated from the same patient.
  • Data shown in Figure 1 illustrates the ability of these PME-CD40L DCs to prime and/or expand a population of T SCM CTLs and also demonstrates that T SCM CTLs can be identified in co-cultures of T-cells and PME-CD40L DCs.
  • T SCM CTLs were identified as shown in Figure 1 by multi-color flow cytometry as cells that are phenotypically CD8+/CD95+/CD28+/CCR7+/CD45RA+. Further testing of these cells revealed that a proportion of the MART-1+ T SCM CTLs were multi-functional, with 1.8% expressing TNF- ⁇ , 3.2% expressing CD107a, and 1.5% expressing IFN- ⁇ .
  • the PME-CD40L DCs contained "GNVR", the RNA antigen payload encoding the antigens GAG (G), Nef (N), VPR (V), and Rev (R). Patients were treated with multiple doses of AGS-004 and the anti-HIV immune response was determined post therapy by monitoring viral load and immune response.
  • PBMCs were collected from the subjects and stimulated in vitro with autologous PME-CD40L DCs encoding HIV antigens from the subject.
  • Multi-color flow cytometry was then used to determine the proliferation capacity and phenotype of stimulated HIV-specific CD4 and CD8 T cells and to identify the activation state of HIV-specific T cells.
  • T cell proliferation was measured by CFSE dye dilution in combination with multi-color flow cytometry.
  • CFSE was a proliferation marker
  • CD27, CD8, CD28, CCR7, CD3, CD45RA, and CD4 were phenotypic markers (see Figure 4 ).
  • Absolute numbers of AGS-004 responding T cells were determined using Trucount tubes. Patients exhibiting proliferating Tscm cells (see Figure 5 ) were selected for comparison of this response with viral load analysis.
  • ART anti-retroviral therapy
  • “Viral rebound” is defined here as the time to detectable virus in the plasma of a patient as measured by standard assays such as, for example, the Roche COBAS® AMPLICOR® HIV-1 MONITOR Test (v1.5, which has a sensitivity of 50 HIV RNA copies/mL) and the Abbott RealTime HIV-1 assay (which has a sensitivity of 40 HIV RNA copies/mL).
  • a delay in viral rebound occurs when the time to detectable virus is longer in a patient compared to other patients and/or compared to the expected time of viral rebound in the absence of effective treatment. Typically, in the absence of any effective anti-HIV treatment, viral rebound would occur within two weeks of ART interruption.
  • the days to detectable viral load is shown in Table 1 below: Table 1: Days to Detectable Viral Load Subject Days 021-002 26 023-001 14 025-004 15 011-009 60 023-002 155 032-001 43 011-005 27
  • additional criteria distinguish a desired clinical outcome.
  • patient 011-005 had almost the same days to detectable viral load as patient 021-002; however, patient 011-005 is considered a good "controller" of HIV because their viral load was much lower and after 12 weeks of ART interruption the patient only had 1690 HIV RNA copies/mL and was able to continue without ART for six additional months.
  • patient 021-002 had a viral load that rebounded to much higher levels (never below 54,600 copies/mL) and had to resume ART after the 12 week assessment period (see, e.g., Figure 6 ).
  • the frequency of CFSElo T cells in a patient represents the percentage of T cells proliferating in vitro after restimulation with the DCs that were transfected with RNA encoding the viral antigens GNVR.
  • Single time point data for the frequency of CFSElo T cells was analyzed for each patient at visit 9 and visit 13 post AGS-004 treatment, but neither time point data showed an association between the percentage of proliferating T cells and extended time to viral rebound in the patient.
  • This proliferating T cell phenotype (i.e., expressing CD27, CD28, and CD45RA) describes T SCM cells for both the CD4 and CD8 T cell populations.
  • FIG 2A , B, C, and D four patients with proliferating CD27 + /CD28 + /CD45RA + CD4 T SCM cells ( Figure 2A ) and CD27 + /CD28 + /CD45RA + CD8 T SCM cells ( Figure 2C ) displayed a longer time to viral rebound and/or decreased viral load after ART interruption (ATI) than three other patients who showed a rapid time to viral rebound.
  • ATI ART interruption
  • the percent change from visit 3 was determined by dividing the mean average percentage of cells in the CD4 ( Figure 2B ) or CD8 ( Figure 2D ) CD27 + /CD28 + /CD45RA + T cell gate determined at visit 9 and visit 13 by the percent of cells in the CD4 or CD8 CD27 + /CD28 + /CD45RA + T cell determined at visit 3 (baseline).
  • AGS-004-induced T SCM cells play a role in the induction of an anti-HIV response in patients that can delay viral rebound during interruption of ART and serve as an indicator of a patient's immune response.
  • patients having cells with greater expression of PD-1 a marker associated with dysfunctional CD4- and CD8-T cell activation, are more likely to exhibit shorter times to viral rebound.
  • decreased PD-1 expression can be an indicator of a successful immune response to HIV.
  • AGS-004 immunotherapeutic intervention may reverse the induction of T cell exhaustion, leading to control of viral replication by inducing long-term immunity.
  • AGS-004 is a personalized dendritic cell (DC) loaded with RNA encoding autologous Gag, Nef, Vpr, and Rev.
  • AGS-004-001 is a Phase 2 trial designed to assess the efficacy and safety of AGS-004 during a 12-week ART structured treatment interruption (ARTI) in chronic HIV-1 infected subjects.
  • This immunotherapeutic intervention is intended to reverse the induction of T cell exhaustion leading to control of viral replication by inducing long-term immunity.
  • AGS-004 consists of matured autologous dendritic cells (PME-CD40L DCs) co-electroporated with in vitro transcribed RNA encoding HIV proteins Gag, Nef, Rev, and Vpr ("GNVR") amplified from participants' pre-ART plasma. These cells are also referred to herein as GNVR DCs or "AGS-004 DC vaccine.”
  • a clinical trial was conducted with HIV patients who initiated Anti-Retroviral Therapy ("ART") within 45 days of acute HIV infection and had HIV RNA ⁇ 50 copies/ml for more than 6 months.
  • AHI was defined as negative or indeterminate EIA or a negative HIV RNA test within 40 days of detectable plasma HIV RNA.
  • Monthly doses of AGS-004 were administered on ART and immune responses ("IR") were assessed after 3-4 doses of AGS-004, at week 12 or 16 of the trial.
  • a positive immune response was defined as an increase in the number of CD28+/CD45RA- CD8+ CTL cells that was two-fold or greater compared to the baseline level and also was 3 standard deviations or more above a negative control.
  • PBMCs were collected from patients pre- and post-treatment with AGS-004 and were cultured in vitro with autologous AGS-004 DC vaccine. After in vitro stimulation, cells were prepared for multi-color flow cytometry and evaluated for expression of surface markers including CD28, CD45RA, CD27, and CCR7 (see Figure 12 ). HIV antigen-reactive CTL subsets were identified and assessed for function, e.g., for the production of cytokines (e.g., IFN-gamma, TNF-alpha, and IL-2); expression of cytolytic markers (e.g. , Granzyme b, CD107); and proliferation (e.g. , as shown by BrdU staining).
  • cytokines e.g., IFN-gamma, TNF-alpha, and IL-2
  • cytolytic markers e.g., Granzyme b, CD107
  • proliferation e.g. , as shown by BrdU staining.
  • Table 2 The demographic and clinical characteristics of these patients are shown in Table 2 below (SCA is Single Copy Assay for HIV; RCI is Resting Cell Infection).
  • Table 3 shows the antigenic response meeting positivity criteria; as indicated in the table, two patients received AGS-004 with only three of Gag, Nef, Vpr, and Rev.
  • Table 2 Demographic and Clinical Characteristics of Patients Receiving AGS-004 DC Therapy for AHI Participant ID Age (years) Race/ethnicity Baseline CD4 count (cells/mm 2 ) Baseline SCA (cps/mL) Length of ATI (days) Reason for ART restart Viral suppression after ATI Baseline frequency of RCI (IUPM) a Post-treatment Frequency of RCI (IUPM) a 51-100 34 African American 662 ⁇ 0.6 36 VL > 10,000 Yes 0.266 0.140 51-102 31 African American 397 -- 268+ b N/A N/A 0.767 0.572 54-100 56 White, non-hispanic 574 ⁇ 0.4 90 VL > 10,000/ >20% ⁇ CD4 Yes 0.179 0.067 54-101 26 White, non-hispanic 482 ⁇ 0.5 147 VL > 10,000 Yes 0.043 0.049 54-102 51 African American 937 ⁇ 0.5 58 >20% ⁇ CD4 Yes c 0.088 0.195 54-104 26
  • ATI Anti-retroviral Treatment Interruption
  • the median duration of ATI was 58 days, with a range of 36 to 147 days, and one patient remained in ATI after 268 days.
  • the baseline SCA Single Copy Assay for HIV
  • FIGS 13 through 18 show multi-functional immune responses ("MIFs") and viral load trajectories for the patients before, during, and after ATI with AGS-004.
  • the figures show absolute numbers of CD28+/CD45RA- CTLs for each marker as well as viral load trajectories.
  • Antigen-specific response for each MIF was determined by subtracting the absolute number of CTLs in the control GFP response plus 3 times the SD from GNVR antigen responses at Week 0 and Week 12.
  • An asterisk above a bar in the bar graph indicates a CTL response that met criteria for positivity, defined in these experiments as at least a 2-fold increase in the absolute number of CTLs for a given (test) antigen determined post-dosing in comparison to the number of CTLs for said antigen per-dosing ( i.e ., at Week 0).

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Claims (9)

  1. Procédé in vitro d'obtention d'une population autologue de cellules enrichies en cellules TSCM aptes à être introduites chez un patient humain comprenant les étapes consistant à :
    a) préparer des cellules dendritiques PME-CD40L matures à partir d'une source tissulaire isolée d'un patient humain ;
    b) cultiver les PBMC obtenues à partir dudit patient in vitro avec lesdites CD PME-CD40L matures dans une coculture pendant une durée suffisante pour induire une augmentation du nombre de cellules TSCM dans ladite coculture ;
    c) enrichir lesdites cellules TSCM à partir de ladite coculture.
  2. Procédé selon la revendication 1, dans lequel lesdites CD PME-CD40L matures sont chargées avec un antigène.
  3. Procédé selon la revendication 2, dans lequel lesdites CD sont chargées avec ledit antigène par transfection avec un ARN codant pour ledit antigène.
  4. Procédé selon la revendication 3, dans lequel ledit ARN est préparé à partir de cellules d'un patient infecté par le VIH.
  5. Procédé selon la revendication 3, dans lequel ledit antigène est préparé à partir de cellules cancéreuses d'un patient atteint de cancer.
  6. Procédé selon la revendication 5, dans lequel lesdites cellules TSCM sont CD8+, CD95+, CD28+, CCR7+ et CD45RA+.
  7. Procédé selon la revendication 1, dans lequel lesdites cellules TSCM sont CD27+, CD28+ et CD45RA+.
  8. Procédé selon la revendication 7, dans lequel lesdites cellules TSCM sont enrichies en tant que cellules positives pour CD27, CD28 et CD45RA.
  9. Procédé selon la revendication 1, comprenant en outre combiner lesdites cellules TSCM avec un support pharmaceutiquement acceptable.
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GB201608060D0 (en) * 2016-05-09 2016-06-22 Univ Birmingham Method of culturing T cells
US10857182B2 (en) 2016-09-23 2020-12-08 Memorial Sloan Kettering Cancer Center Generation and use in adoptive immunotherapy of stem cell-like memory T cells
WO2018072105A1 (fr) * 2016-10-19 2018-04-26 中山大学 Inducteur in vitro de lymphocytes t à mémoire de cellules souches et procédé
WO2018081795A1 (fr) * 2016-10-31 2018-05-03 Zipline Medical, Inc. Systèmes et procédés de surveillance d'une thérapie physique du genou et d'autres articulations
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WO2019231846A1 (fr) * 2018-05-27 2019-12-05 Argos Therapeutics, Inc. Procédés de fabrication de cellules tueuses naturelles et leurs utilisations
KR20210095157A (ko) * 2018-11-08 2021-07-30 넥스이뮨, 인크. 개선된 표현형 속성을 갖는 t 세포 조성물

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AU2015218865B2 (en) 2021-06-10
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